Method for producing high-purity soybean protein



Patented Feb. 27, 1951 I UNITED STATES PATENT OFFICE METHOD FORPRODUCING HIGH-PURITY SOYBEAN PROTEIN Herbert Otto Renner, Des Plaines,111., assignor, by mesne assignments, to J. R. Short Milling Company,Chicago, 111., a corporation of Illinois No Drawing. Application June16, 1947, Serial No. 755,021

15 Claims. (01. 260-1235) 1 2 This invention pertains to new methods ofthe former application, the refined product depreparing high purityproteins. More particusired is item (1) of column 3, Table I, whereas inlarly it relates to the preparation of such prothis applicatio t erefined p duct desired is teins from particulate soybean material (e.g., item (2) of column 3, Table I. flour, meal or flakes) treated withorganic sol-' In t prior a a Vast o t of t me and eX- vents to removesubstances of non-proteinous De have been devoted to efforts to producenature, which cause great difiiculties in the s y-pr t ins free from ntaat puriclarification of protein extracts with resulting ties (larg yn-proteinous in nature), which proteins of unsatisfactory qualit s dis ls d adversely afiect the desirable organoleptic and in my copendingapplication Serial No. 730,539, eep properties of h Proteins and u efiled February 24, 1947, now Patent No. 2,524,991, their suitability ascomponents of plastics and (Case A). The processes constituting thisinother products Where h u y is y vention use soybean products producedby the q yinvention described in my copending application Thediffiolllties d importance Of p odu cited, and hence this application isa continuasoy-pr f h h he p y obtainable tion in part of thatapplication, have been very clearly portrayed by workers in The relationof the invention disclosed in this this field, as for example, by A. A.Horvath in application to that disclosed in my copending ap- The SoybeanIndustry, 1938, pages 147-150. plication Serial No. 730,539, filedFebruary 24, Also, the surprisingly diverse factors for protein 1947, isclearly shown in a general way in Table calculations from nitrogencontent employed by I which appears in that copending application 20previous investigators indicate an unmistakable and is repeated here forconvenience in indicatlack of knowledge in the prior art concerning theing the close relationship of the subject inventrue nature and purity ofthe substances covered tions. by the collective term soybean proteins.Thus,

TABLE I Raw materials used and Methods of using Cellosclves" Refinedproducts aimed at- 'gf figgf g ggggw gg originating in soybeans Ia:Soybean-flakes or-------y(1a) Continuous or- (l) Odor flavorless soy--(l) Cellosolve"-soluble wafiour from whole (i. (1b) batch -extract1 onflakes/meal with imtor-insoluble fractions of e., nonsolvent -e usingvarying ratios Broved properties for raw-material.

tracted) enzyme-acof solids/solvent. uman food.

tive soybeans. (2) Improved soy-flakes! (2) Cellosolve"-soluble wa- Ib:Soybean-flakes or (2) Wetting meal for preparation of tor-solublefractions of flour of la solvent-\ protein of higher purity -'rawmaterial extracted by known (18) Continuous or methods enzyme ac- (lb)batch-extraction (3) Soybean meal oi selec- (3) Cell0solve" insolubletwo or not. tlve enzyme-activity fractions of soy oil (2) Wetting Column1 of Table I shows the raw materials D. B. Jones in 1931 uses a factorof 5.7 (based on used in the processes disclosed in my copending 17.5%nitrogen content of pure glycinine) K. S. application cited and in thisapplication, while Markley and W. H. Goss (Soybean Chemistry columns 2and 3 represent principal flow sheets 45 and Technology, 1 4 P 19) use afactor of of the several methods of processing these raw 2 (based on16.6% n t o en e t of Y- materials in accordance with these inventions.protein); W. G. Smiley and A. K. Smith (Cer. The arrows indicate at aglance the progress of Chem., 1946, page 290) mention as conventionaleach individual method (column 2), with regard factor for determiningthe protein content of to the refined products arrived at (column 3),soybean products avalue of 6.25 (based on 16.0% and the nature of theby-products obtained Nz-content of soy-protein); while Horvath (cited(column 4). The principal difference between above, page 144) advocatesfor use in alkalithe processing of the raw material in my 00- treatedsoybean protein a factor of 6.33 (based on pending application cited andin this case re- Nz-content of 15.8%). sides in the ultimate refinedproduct. Thus, in 55 Accordingly, an object of the present invention isto produce a substantially purer soy-protein than has heretofore beenproduced by methods of the prior art.

Another object of this invention is the production of soy-protein ofincreased purity and yield by the use as starting material of Cellosolverefined soy-meal produced according to the methods disclosed in mycopending application, Serial No. 730,539, filed February 24, 1947.

Another object of this invention is the production of purer soybeanprotein without employing acid-washing prior to alkali-extraction.

Still another object of this invention is to produce higher puritysoy-proteins by two step protein precipitation of soy-proteins fromalkali extracts of Celloso1ve-extracted soy material characterized bythe removal of the precipitate from the whey of the first step beforeraising the pH value of the first step to the pH value required for thesecond step precipitation.

' With these and other objects in view which may be incident to myimprovements, my invention consists in the combination and arrangementof elements and steps hereinafter described and illustrated by typicalexamples.

One of the advantages of using Cellosolveextracted soy material as astarting substance for the production of high purity soy-proteins is thefact that the removal of Cellosolve-soluble fractions from soybean mealresults in a very noticeable increase in nitrogen-content of theresidue, as shown in Table II.

TABLE 11 To clearly distinguish from the prior art and for generalclarity and brevity, certain special terms and abbreviations will beused in this application, with the following definitions:

The term soybean flakes includes any type of soybean particulatematerial obtained by breaking down whole soybeans into flakes, meals andflours of any desired particle size which for practical reasons arepreferably freed from most of their oil by expressing or otherwell-known solvent-extraction methods (such as extraction with hexane).Meal or flakes from whole soybeans, and hydrocarbon(hexane) -extractedsoymeal or flakes (so-called White Flakes of commerce) are both suitableraw materials and yield products of the same basic properties whensubmitted to low temperature-extraction with methyl or ethyl Cellosolve.However, the use of White Flakes as raw material is preferred as itoffers important technical advantages over the use of whole soybeanflakes, as will hereinafter appear.

The. term Cellosolve-refined soybean meal (abbreviated. CRS) is used todenote a soymeal or soy-proteinmeal. prepared by the methods disclosedin my copendin application, Serial No. 730,539, filed February 24, 1947,and possessing all of the properties cited as objects of that invention.

Methyl Cellosolve is abbreviated: m-Cel.

The word Cellosolve, appearing at the to of column 2, Table I andelsewhere in this specifi- C'omparative nitrogen and protein contents ofsoy material extracted by prior art commercial extraction and CeZZosoZoeextraction Protein Nitrogen Equivaiggggf N0. Sample of soy material ondry lent on basis dry basis B Y NIXGDZI Per cent Per cent Per cent 1Commercially extracted 8.54 51.41 6.06

White Flakes." 2 alcohol extracted Soy- 9. 57. 19 6.30

a 'es. Average 1 and 2.- 9.02 54.30 6.18 Methyl-Cellosolve extracted9.80 59. 00 6. 46

ignite Flakes (containing 4 l\Iethyl-Cel1osolve" extracted 10. 27 63. 676. 70

White Flakes (hulls removed by sifting from No. 3). Average 3 and 4--10. 04 61.39 6. 58

the average of factors for protein calculations, as indicated on page 2.

In addition to the foregoing advantage, a study of the properties of theCellosolve-soluble fractions, in particular: their hygroscopicity,watersolubility, low non-proteinous nitrogen, and nonionized phosphoricacid content, and their tendency to form dark colored substances duringstorage in air under normal conditions of humidity and temperature,suggested ver; strongly the probability that if Cellosolve-refined soymaterial is used as a starting substance for the extraction ofcommercial soybean-proteins, it would prove superior to commercialsolvent-extracted soy material (such as White Flakes) heretofore usedfor this purpose. This indication was fully borne out by the results ofmy researches and experiments which form the basis of the presentinvention.

cation, is used for brevity to denote generically the group of alkoxyethanols, comprising methyl Cellosolve (Z-methoxy ethanol or glycolmonomethyl ether), and ethyl "Cellosolve (2-ethoxy ethanol. or glycolmonoethyl ether), in lieu of the longer and more explicit chemicalidentification. While most of the results disclosedin this applicationwere obtained by employing methyl Cellosolve, it was foundthatsubstantially the same results were obtained: bythe use of ethylCellosolve, but the former ispreferred on account of its lower boilingpoint, particularly where low temperature, vacuum distillation orevaporation were the only means permissible for the removal of alltraces of solvent from the product. For the samereason (high boilingpoints) butyl and benzyl Cellosolve, while ordinarily included in thegeneral term Cellosolve, were not used as solvents in the processeshere'- in disclosed and, although they might theoretically be soemployed, their commercial usefulness for this purpose appearsnegligible.

It has been claimed in the literature of the prior art that Washing ofsolvent (hexane)-extracted soybean flakes with acidified. water, priorto extraction with alkali, is effective in retarding the dissolution ofthe globulins and removing those soybean fractions which are consideredthe cause of great difficulties in the clarification of protein extractsand of unsatisfactory quality of soy-proteins precipitated therefrom.However,

ed soybean flakes with acidified water, prior to extraction with alkali,adds materially to the time and cost of producing soy-proteins, it wouldbe very advantageous'to the whole process if this step could be omitted.

In the absence of data from the prior art'concerning the exact acidconcentration, the ratio of soy-flakes to washing water, and theextraction period, it was necessary, in order to demonstrate thesuperiority of Cellosolve-extracted soyflakes over commercial solvent(hexane) -extracted soy-flakes, washed with acidified water, prior toextraction with alkali, to directly compare the results obtained fromCelloso1ve-exno exact information as to the strength of these tractionof White Flake with those obtained acidified waters, nor details ofmethods comfrom washing White Flakes with acidified water merciallyemployed in soy-protein production, are of varying acid (pH)concentrations. Compararevealed in the literature. Indeed, all this datative tests were, therefore, run employing for seems to be a jealouslyguarded trade secret of washing of White Flakes a rather highextracindividual experimenters and manufacturers. tion ratio of 1 partof flakes to 8-10 parts of acidi- There appears to be no single case onrecord fied water, and three concentrations of acid wherein the methodsand sequence of manipula- (acetic), namely: (a) water with a pH of 4.5;tions employed by two workers in this field are (12) water with a pH of3.8; and (0) water conidentically the same, or difier only in a singletaining 0.55% acetic acid and producing wash variable that would permitthe determination of water (extract) showing a pH of 4.5. The esthatvariables influence upon the results 010- sential steps and verysignificant results of the tamed three comparative, parallel tests aregiven in As the washing of commercial solvent-extract- Tables III andIV.

TABLE III Quantities and properties of extractions removed from whiteflakes by washing with acidific water of varying pH, as compared with ICelloso1ve-extraction.]

pH of Wash-Water (with OH OOOH) Table pH 3.8 0.2 cc. pH r 0.55 cc. 1 ees No. DH acid/100 cc. sol.) acid/100 cc. sol.)

B C D Moisture 6.3%.. As A 6.4%. fi i fi gg {Ash (dry basis). 6.19%- an5.94%. Nitrogen (dry basis) 8.67% do 8.41%. }Amount of Flakes {Air-dry100.0 g (in 100.0 g. used Moisture-free 93.7 g rin 93.6 g. Nitrogen,absolute, in #2 8.12 e do 7.87 g. Acidified Water used 1000 cc. 800 cc558 cc. of m-Oe]. Behaviorof Flakes duringwashingonsuetionfilter. Veryconsiderable Swelling same as O o n s i d e r a b Is N o s w e 1 l i n gswelling. filtra- A filtration sl. swelling, filtraspeedy filtrationtion extremely less difficult tion noticeably possible. diificul than inA. easier. 4A Total Washing Time (intentionally prolonged by 300 min 165min 130 min Controllable at filtration). A t 3 725 t 5 moun 691 cc 5 5cc. cc me -ex ract bril- 5A if fl g ggfig g {pH 6.4 5.0-- 4.5." 1mmclear. 5B- Appearance Very turbid Very turbid Turbid Arfiount: As iscontaining 69 g. of A-S 5.9% g. 3-8 7.2%.-" g. 0-5 7.4% 13.4 g. D-S4.1%.

2 6A Solid extractives Amount, on dry basis 64.9 g. A S 41.7 g. B-S 50.0g. C S 12.8 g. D-S. 6B Nitrogen content.

covered from #5 on dI b 0 (in vacuo). 60 (O) Absolutely 0.083 g. 6D (D)In {7520f total N, 1.1%.

o c. Solubil. in M-Oel Not visibly sol. Not visibly sol Not visibly solEasilly completely so 5.4-- 5.4 5.6 5.6 5.5. Solution in Water. pHMilky-white, pre- Very turbid, of- Very turbid Opalescent-Olear. 6E{Properties of Solid cipitatedatpH4.5. fensivc odor.

Extractives #6. Taste Unpleasant, not Highly unpleas- Unpleasant More orless sweetsweetish. ant. ish, unpleasant. Appearance (dry) Brownishyellow Lighter yellow, As B S Yellow-brownish, verybrittle,hymorehygrovery brittle, groscopic. scopicthanA-S. very hygroscopic. 7 Amount,on dry basis 28.8 g. A-R 52.0 g. B-R 42.8 g. OR. 80.8 g. D-R. 7ANitrogen content: 7A Soy Flakes residue (A) On dry basis 8.12% 10.1% 7Bremaining. (B) Absolutely. 2.34 g 6.64 g (O) mtg/60f total N; of 28.8%81.7% v

8 Amount of Soy Fla res Residues #7 containing 118.5 g. A-R 75.4 g. BR..61.9 g. CR.. 100.0 g. D-R or M- same Nr-Amt (9.63%, #7A) as g. ofM-Oel-Gel-extracted extracted Flakes D-R, on dry basis. White Flakes. 8AEquivalent amounts of Air-dry (#2a)-White 411.4 183.4 2 144.3 2 123.7 g.

Flakes yielding the equivalent soy flakes residues of #8.

1 Slightly. Key to abbreviation:

g.=grams. cc.=cubic centimeter. min.=minutes. s=solid extractive. pH=Log(base 10) of the reciprocal of the hydrogen ion concentration in grammolecules per liter. r=residue.

[Precipitability of solid extractives A-S, 13-8 and -8 (Table III, Item#6) in comparison with that of M-Cel-soluble soy fraction D'S.]

Solid extractivcs (Table III, #6)

Table A S B S C S D S A B O D 1 Solid extractives removed from 100 g,air-dry 41.7 g 50.9 g 12.8 g.

White Flakes" on dry basis (Table III, #GA). Total nitrogen-contained in#1, on dry basis 5.78 g 1.48 g 1.46 g 0.083 g.

(Table III, #60). Properties of solutions of extractives #1 in Turbid,dirty Turbid, pure ycl- Same as B "I. V e ry turbid, parts of n/ NaOH.olive green color. low color; on b r o w n 1 s h yelstanding white low;odor very precipitate. much different from that oi B and 0. 2A. Fractioninsoluble in 11/20 NaO None 4.26 e 2.75 g None.

Nitrogen content of #2A:

In per cent 23% N 2.2% No Absolute 0.052 E 0.060 F In per cent of totalN #20 of Table III 64% 0.73% 3 First precipitation with S0; at 110 F. atpH 4.5: AP1:43.47 g B-Pr: 4 05 g CP1:4.40 g S 01. in 4 0 7 solidscentrifuged (1 washing): yield of 1st HCOH D-P precipitate (dried at 63C). 2.68 g. First in per cent of solid extractives #1 20.9%. First inper cent of dry flakes (#ZB, Table III). i 2.3%. Nitrogen content of #3,on dry basis 0.40%. Nitrogen content of #3, absolutely 0.0101 g.Nitrogen content in per cent of total N; in #IA 12.8%. Second treatmentof whey of #3 with excess of Addit onal yellow SO: caused. precipitateD-Pz. Yield of 2nd precipitation; absolute... 3.75 I Yield oi #5A in percent of #1 2 Yield of #SA in per cent of dry flakes #2B, (#2B oi TableIII). Nitrogen content of #5A, on dry basis Nitrogen content of #SA (A)absolutely Ni tlrogen content of #5A (B) in per cent of N2 of n- A.Total precipitates (#3+ #SA) #7 in per cent of total extractivcs #1 8Total nitrogen precipitated: #4A+6 8A- #8 in per cent of total N2 of #lA8B- #8 in tper cent of N; (#20, Table III) wit Key to abbreviation: Sameas Table III. P=precipitate.

In Table III above, columns A to C refer to the before mentioned threeacidified washing waters used while column D, for comparative purposes,shows the results of Washing (extracting) the same white flakes used asstarting material in A to C with m-Cel-solvent. As Table III disclosesall the essential data as to washing procedure and observations madeduring this process, no detailed description is deemed necessary. Themost significant data under #6, and in particular under 611-!) and 6d,prove beyond any doubt the following facts:

1. Washing of white flakes, even with excessive amounts of highlyacidified wash water (columns C and B), fails to remove from solidflakes those extractives removed by m-Cel-extraction (column D) andcharacterized-in contrast to extractives obtained in A to C-by completesolubility in m-Cel (see #66: Solubility in m-Cel). It must, therefore,be concluded that the use of only small amounts of acidified wash waternecessarily will also fail to do so, particularly in view of the factthat due to the extreme swelling and water-imbibing power of whiteflakes (see #4) the amounts of wash water held per 100 g. of whiteflakes (Table III: (#3-#5) in A to C) amounted to 309 cc., 265 cc. and2'75 cc., respectively, and that ratios of flakes to wash waterproteinous nitrogen which would result in extremely uneconomical lossesin contrast to the apparently exclusive removal of non-proteinousnitrogen, proved to be such by the solubility in 40% formaldehyde ofm-Cel-soluble soybean extractives (D-S #6, column D) bym-Cel-extraction. A comparison of figures in item #Ga, columns A to C,with column D shows very strikingly the prohibitive losses in substance,by weight of dry flakes (items #21) and #7), one would encounter inpracticing any of the methods indicated in columns A to C.

3. From paragraphs 1 and 2 above, it follows conclusively that washingof white flakes with acidified water according to methods A to C (whichcover any method possibly used in practice) does not permit theseparation of nonproteinous soybean constituents soluble in m-Cel fromsoy proteins insoluble in m-Cel.

The data of Table IV serve to further fortify the foregoing conclusionsby demonstrating that m-Cel-soluble, non-proteinous, soybean fractions(Table IV, column D and D-S of Table III, #6, column D) containconsiderable quantities Table IV, #8a) of substances precipitable byacid (S02) under conditions under which commercial soybean protein isprecipitated. Thus, soybean protein prepared by prior art methods fromwhite flakes not acid washed before alkali-extraction, of necessity, ismore or less contaminated with non-proteinous impurities forming part ofthe soybean fractions removed from soyflakes by m-Cel-extraction.

The results shown in Table IV are important in several'respects asfollows:

1. The figures under tent of the first precipitate with S02 at pH 4.5,plainly indicate that the precipitate D-Pl (#3, column D) with aNz-content of 0.40% differs fundamentally from the correspondingprecipitates A-P1, B-P1 and C-P1 obtained from extractives A-S, 13-6 andOS, respectively,'while the closeness of these Nz-values under #4 for A,B, and C indicates that these precipitates contain nearly equalpercentages of the same protein contaminated with nearly equalpercentages of non-proteinous precipitate of the type D-P1 of column D.Similarly, impure precipitates are undoubtedly obtained when soybeanproteins are precipitated, by processes disclosed by the prior art, fromalkali-extracts of soy flakes'not previously extracted with m-Cel.

2. The figures in Table IV under #5 to #6?) reveal that, due to thedifferences in chemical properties between the m-Cel-soluble soybeanextractives (D-S) and the extractives (A-S, B-S and C-S), theprecipitability of D-S is increased by considerable lowering the pHbelow 4.5 while the precipitability for A-S, 3-8, and C-S had alreadyreached its maximum at pH 4.5 (first precipitation). The figures under#8b indicate that the precipitable non-proteinous soybean fractionscontained in D-S, D-P1 and D-Pzcan exert their influence upon theNz-content of precipitates such as A-P1, 'B-P1 and C-P1 only by loweringthe latters nitrogen content by dilution TABLE V #4, as to nitrogencon-..

by m-Cel-extraction obviously not only raises the Nz-content of thesoybeans, as 'shownby Table II above, but also increases the N2 (orprotein)- content of soybean proteins precipitated from alkali extractsof m-Cel-extracted soymealsr' 10 The conclusions of the foregoingparagraph (3) are further supported by the experimental evidence shownin Table V which represent the results of four series -(-S1,-S2, -S3--and S -4)--of comparative protein-extraction and precipitation 5experiments using, as starting material commercial hexane-extractedsoyfiakes, not m-Cel extracted as well as m-Cel-extracted. The 'methodsof alkali-extraction and protein precipitation employed in the fourseries, have been intentionally varied to cover a great number ofvariations which commercial practice could possibly use, but it isexpressly understood that painstaking care was taken to avoidany'variations in the manipulations within each individual series tomake the results as comparative as possible, and in no case was anattempt made to exhaustively extract all alkali-soluble matter from theraw materials (S) started out with. For comparative purposes, most ofthe data in Table V are calculated on the basis of grams ofmoisture-free (dry) soybean raw material started out with.

Typical comparative analyses of I. Soybean raw materials (S) for proteinextraction; f II. Acid-precipitated protein (P) from alkali-extracts of(S); and III. Soybean residues (R) from protein extraction of (S) byalkali showing the influence of 3 m-CeZ-extraction of (S) upon thecomposition of the respective (P) and (R) I. SOYBEANRAW MATERIALS (5)FOR PROTEIN EXTRACTION S-l S-lA S-2 S-2A S-3' Tg ble I o. White Flakes IM -Cel Extr. S1 White Flakes M-Oel Extr. S-2 Same as S 2 100 gramscontain: 1 Dry matter. 93.6 P 93.2 2 92.6 2 91.9 E 926g; 2 1-. 7.87 P8.77 9 7.98 g 9.10 7.98 g. 3 100 grams dry matter contain per cent8.41%.... 9.72%.. 8.61%.. 9.90% 8.61% N: or g N2.

S4 H $4.13 -fif-q. Tlgble V V Alcohol Extr. Same as S 2A White Flakes MOel Extr. S 4 Same as S 1A soyflak-esm 100 grams contain:

Dry matter-.. 91.9 g 93.7 G 93.6 g 93.2 v 93.3 g. N2... 9.10 g 8.12 g9.17 E 8.77 g 8.91 g. 100 grams dry matter contain per cent 9.90%.8.67%. 9.80%. 9.72% 9.55%.- N z or g N a.

TABLE V-Continue (1 Typical comparative analyses of I. Soybean rawmaterials (S)v for protein extraction;

II. Acid-precipitated protein (P) from alkali-extracts of (S) and III.Soybean residues (R) from protein extraction of (S) by alkali showingthe influence of m-CeZ-erctraction of (S) upon the composition of therespective (P) and (R) 11. ACID-PREOIPITATED PROTEIN (P) FROMALKALI-EXTRACTS OF s) S-l S-IA S-2 S-2A S-(l TNa ble White Flakes M-OelExtr. S-1 White Flakes M-Cel Extr. S2 Same as 8-2 Extraction-Ratio". 4 1(g air-dry S) A (cc. NaOH Sol'n) 1/20 1/20. 1/15 1/15 1/15. 4A Extractcontaining 13% NaOH 0.1% 0.1% 0.15% pH 0.15% pH 10 0.15% pH 10. 413 1stExtract-period 30 min 30 min 1 hr. (77 F.) 1 br. (77 F.) 1- hr. (77 F.).

Total. extract. (from all extractions in.- cluding wash-water)centrifuged: 4C Cc. per dry matter #1 1350 ec 1350 cc. 1487 cc 1395 cc1403.

(1 extraction (1 extraction). 8-1 wash 1:1). (1 wash 1:1}5). 1442 cc-1448 cc 1606 ce 1518 cc 1450 cc.

4D 00. per 100 g dry S (1 extraction N o washing) 4E Solids content per100 g dry S 74.17111; (see table 62.1 g 63.8 g.

4F Other properties Darker color. Lighter color, More intense Lightbrownish Considerably more turbid less turbid colored than yellow.darker more than S-lA. than 5-1. in S-ZA. 1 tSu3rAbid than 5 1stPrecipitation:

5A Acid used H01 H01 SOi-gas.. SOi-gas SOs-gas.

At Temp; Room Temp. Room Temp. 39 0. (104 F.) 30 0. (104 F.) 30 0. (104F.).

(75 F.). (75 F.). At pH- 4.8.-- 4.5.-- 4.5... 4.5

(4.4+NaOH).

Yield of filtered Wiley 1 (See #6) 1371 cc.

Solids content of whey 1 25.2 g.

Yield of filtered protein wet I Yield of protein air-dry 1 31.0 g 29.0 g39.4 g.

Per cent H in #56 6.04% 6.50%.- 10.3%.-. 6.5%.-- 5.80%.

Dry matter in #5 G 27.8 i 27.1 g-.- 37.1 g.

Per cent N2 111 #51 (-g Nz) 15.37% 16.44% 13.96% (3.88 g.) 15.23% (4.13g.) 14.71% (5.45 g.).

#5J in per cent of N2 of 45.0 41.6%.. 63.2

Other properties of protein Dark brown, Oonsiderably Darker brown, moregummy lighter col r more opaque in air-dry state than 5-2; not than,notiransthan S-2A. brownish; parent glassmore glassy- 1 like as S-3A.brittle. 2nd Precipitation:

From #5D by raising pH to At temperature Yield of filtered protein; wet

(pressed dry).

Per cent- H2O in 60.....

Dry matter in #60, g

N1 in #60, grams, er cent.

Other properties of protein..-

Total precipitates (air-dry): #5G 0. 31.0 c 29.0 39. g. Total Nrin. #7inper cent oi. N1. oi #3 45.0%-. 41.6%- 63.2%.

(sK-i-s S-3A S-4 S-4A 8-413 8-40 Table Same as 8-2. White Flakes M-CelExtr. S-4 Same as S-IA gggl ig Extraction-Ratio: 4 1 (g air-dry S) A(co. NaOH Soln) 1/15 1/ 1/15. 4A -Extractcontaining B-% NaOH' 0;125%0.125 0.125%. 4B 1st Extract-period hr, (77 F 1 hr, (82 F,) 1 hr. (82F.).

Total extract (from all extractions ineluding wash-water) centrifuged:4C Cc. er dry matter #1 1165 cc 1810'cc- 1785 cc.

- (2 extractions (2 extractions (2 extractions) (1 and 1 wash no wash).wash). 1:2

cp 00 g. y S 1267 cc 1031 cc 1858 cc 1013 cc.

Solids contentper 100 g. dry S 48.0 g 68.3 g 58.9 g 05.5 g.

other P D Much lighter, Dark brownish, Light greenish, More turbid thannot turbid as very turbid opalescent. S4A. m u c 11 8-3. more 0010 re dt h a n 8-4.4, cleaner tli a 11 8-4.

1st Precipitation:

Acid used SOz-gaS SO -gas SOi-gas SOi-gas.... 801-1205.

At p-" 39 O. (104 F.) 0. (104 F.) 39 0. (104 11).- 39 0. (104 F.) 30 0.(104 1 DH 4.6 (4.4+N11OH) 4.5 4-5 4.5.

Yield of filtered whey 1 (see #6) 1371 cc 1691 cc 1682 cc 1609 cc 15 cc.

Solids content of whey 1 21.2 g 38.3 H 26.4 g 20.6 g 34.1 g.

Y eld o1 filtered protein wet l 274 g 210 g.. 236 g. 303 g.

ield 01' protein air-dry 32.6 c 34.7 g 33.1 g 32.1 g 34.3 1:.

Per cent H19 in #5G 5.80% 6.0% 4.9% 8.4% 12.5%.

Dry matter in #5G 30.7 g 32.6 g 31.5 g. 29.4 o 30.0 g.

1 Per g. of dry S.

TABLE V-Continued Typical comparative analyses of I. Soybean rawmaterials (S) for protein extraction; II. Acid-precipitated protein (P)from alkali-extracts of (S); and III. Soybean residues (R) from proteinextraction of (S) by alkali showing the influence of m-C'el-erctractionof (S) upon the composition of the respective (P) and (R) n.AOID-PREOIPITATED PROTEIN (P) FROM ALKALI-EXTRAOTS or (S)ontinued S-3AS-4 S-4A S-4B S- Table Same as S-2A White Flakes M-Cel Extr. S-4 Same asS1A Alcohol Extr.

soyliakes Per cent Na in #5i (-g NZ) 15.38% (4.72 g.). 13.87% (4.53 g.)15.38% (4.84 g.) 14.97% (4.40 g.)-- 14.08% (4.22 g.).

#51 in per cent of N2 of #3- 47.6%... 52.2%.- 49.4%.. 44.2%.

Other properties of protein Considerably Decidely brown- V e r y m u c hAbout as S-4A.- Decidedly olivelighter, pure ish-yellow collighter thangreenish less yellow color or, opaque. S-4, color puryellow than S t h an S 3 er, more trans- 4A, less browng1 a s s l i k e parent than ishthan 84. transparent. S4 and S-40.

2nd Precipitation:

6 From #5D by raising pH to.-.---- 4.8- 9 8.

6A At temperature.. 104 F 6B----. Yield of filtered protein; wet 36.2 g.creamy (pressed dry). yellow. hite.

6C -Air-dry 11.2 Q

6D Per cent H1O in 6c..... 9.6%.. .9

6E Dry matter in #60, Q 10.1 e 6.8 e 8.9 H 11.9 g.

6F N1 in #60, grams, per cent....---- 1.65 g., 16.36%--- 1.12 g.,16.50%... 1.43 g., 16 13% 1.86 g., 15.63%.

#(SF in per cent of N2 of #3 19.0%.. 11.4%.. 19.4%.

GB Other properties of protein Glassy mass, Without pure Lightest colorof considerably yellow color 2nd precipitate darker than of 1stprecipiof S-4, -4A, 1st precipitate. tate. 40.

7 Total pecipitates (air-dry): #5 G+#60. 32.6 e 45.9 g 40.5 a 42.2 g47.1 g

7.4 Total 2 in #7 in per cent of N2 of #3 47.6%.... 71.2%. 60.8%.. 59.9%63.6%.

III. SOYBEAN-RESIDUES (R) FROM PROTEIN-EXTRACTION OF (S) A000 RDING TOII S-l S-IA S-2 S-2A S-3 Tlgble White Flakes M-Cel Extr. S-1 WhiteFlakes M-Ce] Extr. S2 Same as S-2 Soy-residue from #5D, pressed 011 perg. of dry S. Water lost during drying of #8 of F. Residtue #8, air-dry:HzO-content: Per 45.3, 9.0%.

cen Residue #8 on dry basis.. 41.2 g. N: in #80, in per cent 5.12% #8Din per cent of N2 of 24.5%

Total N2 recovered in #7A-i-8E in per 87.7%.

cent of #3.

S-3A S-4 S-4A S-4B S-40 Tlgble Same as S-2A White Flakes M-0el Extr. 84Same as S-lA gg ggg g Soy-residue from #5D, pressed off per 205 g-. 269g. 276 g 294 g.

100 g. of dry S. Water lost during drying of #8 of 120 F. 164.4 218.8 Q231.0 1! 251.2 g.

Residue #8, air-dryz HzO-COlltBlltZ Per 56.0 g 7.2%.--.- 40.5 g.,9.7%...- 50.2 g., 9.9%..--. 45.0 g., 6.9%.-.-- 42.8 g., 6.2%.

ccn Residue #8 on dry basis 36.6 g 45.2.. N: in #80. in per cent.-4.82%-. 6.58%.- #8D in per cent of N: of 20.3%.. 30.4%.. Total N;recovered in #7A 91.5%-- 91.2%...

cent of #3. r

Per 100 g. of dry S.

which, by extraction with methyl-Cellosolve,

the respective m-Cel extracted soyfiakes S-la,

S-Za, S-3a. and S-4a have been prepared (S-4b is the same as S-la, whileS4c is an alcohol extracted soyfiake). Horizontal lines 1 to 3 giveessential analytical data concerning these raw materials for soy proteinproduction.

Part II of Table V discloses in horizontal lines 4a to 4] thecomparative preparation of alkali extracts of the above raw materials bymethods differing by: extraction ratios (line 4); NaOH- concentration ofextracts (line 4a); and extracin each case the resulting influenceswhich the above variations in extraction procedure exert upon theamounts of extracts obtainable (lines lo-4d), and the latterssolid-content (line 4e), and colors (columnj).

These alkali extracts were generally prepared by immersing, for example,with an extraction ratio of 1/20 (one unit of weight of the soy materialin 20 units of volume of alkali solution), gently agitating for aspecified time, removing the coarse solids by screening and the fines bycentrifuging.

In Part II of Table V, horizontal lines 5a to 5Z illustrate theinfluence which difierences of soy tion periods (line 4b) While lines 4oto 4f show 5 bean rawmaterialsujsed for protein production exert uponthe properties of proteins precipitated from the above alkali extractsby carefully acidifying the latter with acids (line a) at certaintemperatures (line 55) to a certain pH (line 50). The precipitatedprotein is removed by centrifuging. Part III, of Table V shows thesoybean residues (R) which result from protein-extraction of soyflakes,according to the methods indicated in Part II of Table V.

The most important results are those under #5 which show that no matterwhat kind of extraction method and precipitation procedure (with pHvarying between 4.5 and 4.8) was employed, the nitrogen content of theprecipitated proteins obtained from the m-Cel-extracted soyraw materialsappeared to be consistently and substantially higher than that ofproteins obtained from non-m-Cel-extracted soyflakes. The Nz-increases(1.07%; 1.27%; 0.67%; 1.51%; 1.10%) averaged 1.12 including the resultof S-3 vs. S-3a-series which because of certain experimentaldifliculties during protein-precipitation (see #50), with a differenceof only 0.67% fell out of line. The 0.21% result of 8-4 vs. S-4c, with asample of ethyl alcohol-extracted soyflakes, demonstrates, in comparisonwith S-4a and S-4b, that alcohol-extraction of soyflakes does notaccomplish what m-Cel-extraction does. It should be borne in mind thatthe above 1.12% average-nitrogen increase is the equivalent of 6.74% (N6.02) to 7.00% (N 6.25) of protein, discussed hereinbefore.

As the data of Table V, #59 (yield of air-dry protein), #52 (yield ofdry protein) and #7 (total air-dry precipitates) indicate, no increasein the yield of proteins precipitated under the experimental conditionsof the tests was observed with m-Cel-extracted soyflakes, said proteinyield apparently being greatly dependent upon the thoroughness withwhich the soy-raw materials were extracted by alkali. The figures ofTable V, #80 (soy-residues from extractions on dry basis) and 8e(nitrogen in #80 in percent of N2 of #3) illustrate very plainly that inall cases m-Cel-extracted soy-raw materials have been less exhaustivelyextracted under equal conditions than non-m-Cel-extracted soy-materialwith the result that nitrogen contents of dry extracted soy-residues(#Sd) from m-Cel-extracted raw materials are very considerably higher(8-4 vs. S-4a:1.76%, S4 vs. S-4b: 2.00%, S-3 vs. S-3a: 1.66%; 8-2 vs.S-2a: 2.91%: 8-1 vs. S1a: 1.15%) than those of the correspondingsoy-materials not extracted with m-Cel.

Alkali-extraction of the m-Cel-extracted raw materials, until theNz-contents of the residues (#Sd, Table V) and the degree ofNz-extraction from the raw materials (#83, Table V) reach the valuesobtained in corresponding extractions of non-m-Cel-extracted soymaterials, increase the yield of higher quality soy proteins fromm-Celextracted soyflakes above that normally obtained with ordinaryextracted soyflakes.

The correctness of this highly important conclusion appears strikinglyillustrated by the results of a comparative extraction and precipitationtest of soybean raw materials S-1 and S-la of Table V, made bydispersing 300 g. of air-dry flakes in 3000 cc. of water of 27 C. (306F.), raising the pH to 10 with NaOH, filtering through cheese clothafter one hours stirring, washing of residue with 100 cc. water,centrifuging in 250 cc. centrifuge tubes, acidifying the (2600 g.)filtrate with SOz-gas to pH 4.5, after having raised the temperature to39 C. (104 F), allowing to stand allnight, filtering and air-drying thefiltered off material, yields of 66.5 g. for S-1 and 84.5 g. for S-la,respectively, were obtained. Thus, the yields of air-dry precipitates,per 100 g. of airdry flakes started out with, amounted to 22.16 g. and28.16 g., respectively and the yield from S-la surpassed that from S-lby 6.0% by weight of air-dry raw material or 27.0% by weight of theprecipitate from S-l.

Further proof that alkali extracts from m-Celextracted soy materials arepurer than those from ordinary white flakes is given by the figures ofTable V (#5e) which show that the solidscontent of the whey from whichthe first precipitate has been removed is noticeably lower wheneverm-Cel-extracted soyflakes were used as raw material. At the same time,figures of Table V (#6e and #Gg) indicate that the amount of substancescontained in the said whey from the first precipitation, andprecipitable at a pH of 4.8-4.9, is noticeably smaller wheneverm-Celextracted flakes are used as raw material (S-4a, S-4b) In otherwords, in the latter cases noticeably smaller losses (compare Table V,#69: S-4a and S45 vs. S4) of proteinous precipitable matter, notprecipitated at pH 4.5, would be encountered under the conditions of theabove experimentsand the Nz-contents (Table V, #67), of all of the 2ndprecipitates of 8-4, 4a and -4c indicate that the latter arerepresenting practically pure proteins when the most widely usedconversion factors 6.02 to 6.25 are used. The increased yields ofprecipitable nitrogenous compounds obtained by following up a firstprecipitation at pH 4.5 by a second precipitation at pH 4.8-4.9 suggesta similar procedure as a commercial means of further increasing thepurity and yield of soy proteins.

The beneficial effect of the removal of the m-Cel-soluble fractions fromsoybean-material follows from the colloidal nature of the saidm-Cel-soluble fractions which adversely affect the particle size of theprecipitates, and the rate of sedimentation and filtrability of suchprecipitates from dispersions, and which are believed to be largelyresponsible for the difficulties heretofore encountered and overcome bythe methods disclosed herein.

The non-proteinous nature of the m-Cel-soluble soybean-fractions, makesit obvious that soyprotein originating in m-Cel-extracted soybeanrawmaterial is superior to protein from ordinary soybean-material withregard to its suitability as a component of plastics and other productswhere protein-purity is the key to quality.

The essential steps of the above described novel process for producingproteins of higher purity may be summarized as follows.

Hexane extracted soyflakes, commercially, known as white flakes areextracted with methyl Cellosolve according to the methods described inmy copending application, Serial No. 730,539, filed February 24, 1947(Case A); the methyl Cellosolve is removed from the flakes by the usualmethods, preferably including a vacuum treatment to avoid raising thetemperature of the flakes; these extracted flakes are then furtherextracted in an aqueous solution of pH varying between 7 and 11(preferable pH of approximately 10); the residual flakes are thenremoved by centrifuging or screening, and the solution is acidified withany preferred acid, such as hydrochloric or sulphuric acid, or withgaseous $02, at a pH, near the iso-electric point of the soya-beanprotein, or approximately 4.5; the prel? 'c'ipitated protein is removedfrom the whey the pH of which is then raised to 4.8-4.9 producing asecond precipitation of protein; the precipitated proteins are separatedfrom the liquids by centrifuging or other mechanical means and dried.The characteristics of the resulting proteins can, of course, be changedby variations in. the time and temperature of the alkaline treatment andtemperature of precipitation, as indicated in Table V above.

The invention disclosed herein has been described for illustrativepurposes in its preferred embodiment, but it is to be understood thatthe scope of the invention is defined by the appended claims rather thanby the foregoing description.

What I claim is:

1. A process of producing high purity proteins which process comprisesextracting oil-free soybean flakes with an alkoxy ethanol, removing theextract from the residue, extracting the residual flakes in an aqueoussolution of pH varying between 7 and 11 and removing the residue,acidifying the resulting extract, removing the resulting precipitatedprotein from the whey, and drying said protein.

2. A process of producing high purity protein which process comprisesextracting hexane-ex tracted soybean. flakes with methoxy ethanol,removing the extract from the residue, extracting the residual flakes inaqueous solution of pH varying from 7 to 11 and removing the residue,acidifying the resulting extract with gaseous sulphur dioxide to a pH ofapproximately 4.5, removing the resulting precipitated protein from thewhey, raising the pH of said whey to 4.8 to 4.9 thus producing a secondprecipitation of protein, separating said precipitate from its Whey, andcombining and drying the precipitated proteins.

3. A process of producing high purity proteins which process comprisesextracting hexane-extracted soybean flakes with an alkoxy ethanol,removing the extract from the residue by draining and vacuum treatmentwithout raising the temperature of said flakes, extracting the residualflakes in an aqueous solution of pH varying between 7 and 11 andremoving the residue, acidifying the resulting extract, removing theresulting precipitated protein from the whey, and drying said protein.

4. A process of producing high purity proteins which process comprisesextracting hexane-extracted soybean flakes with an alkoxy ethanol,removing the extract from the residue, extracting the residual flaflesin an aqueous solution of pH varying between 7 and 11, and removing theresidue, acidifying the resulting extract, removing the resultingprecipitated protein from the Whey, and drying said protein.

5. A process of producing high purity proteins which process comprisesextracting hexane-extracted soybean flakes with an alkoxy ethanol,removing the extract from the residue, extracting the residual flakes inan aqueous solution of pH approximately 10, and removing the residue,acidifying the resulting extract, removing the resulting precipitatedprotein from the whey, and drying said protein.

6. A process of producing high purity proteins which process comprisesextracting oil-free soybean flakes with an alkoxy ethanol, removing theextract from the residue, extracting the residual flakes in an aqueoussolution of pH approximately and removing the residue, acidifying theresulting extract to a pH of approximately 4.5,

18.. removing the resulting precipitated protein from the whey, anddrying said protein. v

7. A process of producing high purity proteins which process comprisesextracting oil-free soybean flakes. with an alkoxy ethanol, removing theextract from the residue, extracting the residual flakes in an aqueoussolution of pH approximately 10 and removing the residue, acidifying theresulting extract with hydrochloric acid, removing the resultingprecipitated protein from the whey, and drying said protein.

8. A process of producing high purityproteins which process comprisesextracting hexane-extracted soybean flakes with an alkoxy ethanol,removing the extract from the residue, extracting the residual flakesin. an aqueous solution of pH approximately 10 and removing the residue,acidifylng the resulting extract. with sulphuric acid, removing theresulting precipitated protein from the whey, and drying said protein.

9. A process of producing high purity proteins which process comprisesextracting hexane-extracted soybean flakes with an alkoxy-ethanol,removing the extract from the residue, extracting the residual flakes inan aqueous solution of pH approximately 10 and removing the residue,acidifying the resulting extract with gaseous sulphur dioxide, removingthe resulting precipitated protein from the whey, and drying saidprotein.

10. A process of producing high purity protein which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol,removing the extract from the residue, extracting the residual flakes inaqueous solution of pH approximately 10 and removing the residue,acidifying the resulting extract to a pH of approximately 4.5, removingthe resulting precipitated protein from the whey, raising the pH of saidwhey to 4.8 to 4.9 thus producing a second precipitation of protein,separating said second precipitate from its whey, and combining anddrying the precipitated proteins.

11. A process of producing high purity proteins which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol,removing the extract from the residue by vacuum treatment withoutraising the temperature of said flakes, extracting the residual flakesin an aqueous solution of pH approximately 10 and removing the residue,acidifying the resulting extract to a pH of approximately 4.5, removingthe resulting precipitated protein from the whey, raising the pH of saidwhey to 4.8 to 4.9 thus producing a second precipitation of protein,separating said second precipitate from its whey, and combining anddrying the precipitated proteins.

12. A process of producing high purity protein which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol,removing the extract from the residue, extracting the residual flakes inaqueous solution of pH varying between 7 and 11, and removing theresidue, acidifying the resulting extract to a pH of approximately 4.5,removing the resulting precipitated protein from the whey, raising thepH of said whey to 4.8 to 4.9 thus producing a second precipitation ofprotein, separating said second precipitate from its whey, and combiningand drying the precipitated proteins.

13. A process of producing high purity protein which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol.removing the extract from the residue, extracting the residual flakes inaqueous solution of pH 76 approximately 10, and removing the residue,

acidifying the resulting extract to a pH of approximately 4.5, removingthe resulting precipitated protein from the whey, raising the pH of saidwhey to 4.8 to 4.9 thus producing a second precipitation of protein,separating said second precipitate from its whey, and combining anddrying the precipitated proteins.

14. A process of producing high purity protein which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol,removing the extract from the residue, extracting the residual flakes inaqueous solution of pH varying from 7 to 11 and removing the residue,acidifying the resulting extract with hydrochloric acid to a pH ofapproximately 4.5, removing the resulting precipitated protein from thewhey, raising the pH of said whey to 4.8 to 4.9 thus producing a secondprecipitation of protein, separating said second precipitate from itswhey, and combining and drying the precipitated proteins.

15. A process of producing high purity protein which process comprisesextracting hexane-extracted soybean flakes with methoxy ethanol,removing the extract from the residue, extracting 20 the residual flakesin aqueous solution of pH varying from 7 to 11 and removing the residue,acidifying the resulting extract with sulphuric acid to a pH ofapproximately 4.5, removing the resulting precipitated protein from thewhey, raising the pH of said whey to 4.8 to 4.9 thus producing a secondprecipitation of protein, separating said second precipitate from itswhey, and combining and drying the precipitated proteins.

HERBERT OTTO RENNER.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 1,260,656 Bollman Mar. 26, 19182,191,455 Davis Feb. 27, 1940 2,200,391 Freeman May 14, 1940 2,233,213Kniseley et a1 Feb. 25, 1941 2,278,670 Rauer Apr. 7, 1942 2,354,393Manley et a1. July 25, 1944 2,405,830 Irving et a1 Aug. 13, 1946

1. A PROCESS OF PRODUCING HIGH PURITY PROTEINS WHICH PROCESS COMPRISES EXTRACTING OIL-FREE SOYBEAN FLAKES WITH AN ALKOXY ETHANOL, REMOVING THE EXTRACT FROM THE RESIDUE, EXTRACTING THE RESIDUAL FLAKES IN AN AQUEOUS SOLUTION OF PH VARYING BETWEEN 7 AND 11 AND REMOVING THE RESIDUE, ACIDIFYING THE RESULTING EXTRACT, REMOVING THE RSULTING PRECIPITATED PROTEIN FROM THE WHEY, AND DRYING SAID PROTEIN. 